Project Summary The focus of Project 2 of this Program Project is to understand the molecular basis for the restriction of cultivation of human noroviruses (HuNoVs) in cell systems. The inability to cultivate human noroviruses is the largest barrier to understanding norovirus biology, transmission and pathogenesis. The clinical and economic significance of HuNoVs as the most common cause of epidemic and sporadic cases of acute gastroenteritis worldwide is now well documented, including their being the most important cause of global foodborne and waterborne illness. In spite of their impact on human health, the underlying mechanisms of pathogenesis and factors regulating virus replication, persistence and transmission are poorly understood. This lack of understanding and the inability to develop measures to inactivate or neutralize virus is largely due to the lack of available in vitro cultivation or robust animal models of infection. Our prior research shows HuNoVs grow readily in genetically susceptible hosts. We hypothesize that virus replication is regulated by a still unidentified co-receptor for viral entry into cells, by viral proteins or by cellular innate responses and signaling pathways that restrict HuNoV replication in cultured cells. Identification and infection of the correct, nontransformed human target cell is also necessary. Our overall goal is to exploit fundamental discoveries as well as new tools developed in the previous project period that produce reporter-tagged HuNoV to obtain a molecular understanding of host and viral interactions that control virus replication. In Specific Aim 1, we will use new nontransformed, ex-vivo complex 3D human intestinal ?mini-gut? cultures to cultivate HuNoVs and biopsies from patients with chronic disease to identify the cell types infected by HuNoVs. We will obtain a variety of HuNoV strains including quasispecies from samples from immunocompromised patients for cultivation. In Specific Aim 2, we seek to understand the molecular mechanisms by which norovirus protein expression regulates cellular responses and how cellular responses regulate viral replication and spread. We will examine the role of IFN? in HuNoV RNA-transfected cells by inhibiting key modulators in the IFN? pathway to determine whether IFN? affects viral replication and spread. We also will also use an unbiased screen to determine the breadth of cellular responses elicited by HuNoV RNA replication in cells and identified key cellular pathways that may restrict virus replication will be evaluated. Additional studies will genetically manipulate HuNoV genes to dissect protein function. The focus will be to probe the structure function of protease and on p22 and p41 NTPase with Project 3, to understand their roles in membrane rearrangements and regulation of cellular protein secretion. Overall, these studies are designed to lead to a fully permissive replication system, gain new molecular understanding of host and viral functions that control virus replication and provide new insight about the pathophysiology of disease.